Chimeric Proteins To Enhance Crystal Formation Of G Protein Coupled Receptors. This R21 proposal is in response to the Program Announcement on Membrane Protein Production and Structure Determination (RFA Number: RFA-RM-04-026). The overall goal of this proposal is to develop a general approach to obtain high-resolution structures of G protein coupled receptors (GPCRs) that can be used for structure-based drug development. The majority of hormones and neuretransmitters communicate information to cells via GPCRs. GPCRs represent the largest family of membrane proteins in the human genome and the largest group of targets for drug development. We propose to use the human beta2AR as a model system to develop a method for generating diffraction quality crystals of GPCRs by increasing their stability and their hydrophilic (polar) surface area. This will be accomplished by generating structurally integrated chimeric proteins in which a soluble protein is integrated into a GPCR by replacing the third intracellular loop (ICL3) and the carboxyl terminus. This approach will address two important obstacles in the generation of GPCR crystals: (1) The ICL3 and carboxyl termini of GPCRs are the most dynamic (flexible) and unstructured domains in most GPCRs. These domains will be replaced by a highly structured homogeneous protein. The three splice sites will ensure a rigid structural coupling between the soluble protein and the GPCR. (2) The soluble protein component of the chimera will significantly increase the polar surface area available for crystal lattice contacts. If successful with the beta2AR, this approach will likely be generalizable to other GPCRs. Specific aims include: Aim 1. Use molecular modeling tools to identify soluble proteins that can be fused to the beta2AR to generate stable, functional chimeras. Aim 2. Generate, express and optimize fusion proteins. Lay summary. The goal of this proposal is to develop a general method to obtain high-resolution structures of G protein coupled receptors (GPCRs). These structures should facilitate the process of drug discovery for GPCRs, which are the largest family of membrane proteins in the human genome. Drugs acting on GPCRs can have an impact on a broad spectrum of diseases including: cardiovascular disease, pulmonary disease, inflammation, diabetes and obesity, behavioral disorders and Alzheimer's disease.